ABC transporter proteins represent a large superfamily of proteins with conserved features in both prokaryotes and eukaryotes. ABC transporters catalyze ATP-dependent transport of endogenous or exogenous substrates across biological membranes (Borst, P., (1997) Seminar in Cancer Biology 8:131-213) and/or allosterically modify the function of heterologous proteins (Higgins C F, 1995, Cell 82:693-696). Several ABC transporters have been associated with clinically relevant phenotypes including the phenomenon of multidrug resistance (Ambudkar S. V. et al., (1999), Annu. Rev. Toxicol., 39:361-398), cystic fibrosis (Riordin J R et al., (1989) Science 245:1066-1073), atherosclerosis (Brooks-Wilson A et al., (1999) Nature Genetics 22:336-345), hyperinsulinemic hypoglycemia (Thomas P M et al., (1995) Science 268:46-429), macular degeneration (Allikmets R et al., (1997) Science 277:1805-1807), and adrenoleukodystrophy (Mosser J et al., (1993) Nature 361:726-730) to name but a few. While the genes associated with these disease phenotypes have been identified to some degree, it is clear that a large number of putative ABC transporters exist in the human genome, as evidenced by the partial sequences noted in various EST databases (Allikmets et al., (1996) Hum Mol Genet 5:1649-1655). However, the utility of such information is compromised by the absence of the full-length nucleotide sequence of the coding region of the relevant gene and its translated protein product.
The present invention is based, at least in part, on the discovery of a novel ATP Binding Cassette (ABC) transporter family member, referred to herein as ABCB9 transporter nucleic acid and protein molecules The ABCB9 transporter molecules of the present invention are useful as targets for developing modulating agents to regulate a variety of cellular processes, particularly the transport of neurotoxic molecules, e.g., xcex2-amyloid peptide (Axcex2), across cell membranes or, e.g., the blood-brain barrier (BBB). Neurotoxic molecules such as xcex2-amyloid peptide are involved in neurological disorders such as Alzheimer""s disease (see, e.g., Goate et al. (1991) Nature 349:704; Games et al. (1995) Nature 373:523; and Suzuki et al. (1994) Science 264:1336). Other neurological diseases involving toxic polypeptides include, e.g., prion diseases, Huntington""s disease, Parkinson""s disease, Amyotrophic Lateral Sclerosis, Spinocerebellar Ataxia, Frontotemporal Dementia, etc. (Hardy et al. (1998) Science 282:1075-1079; Wolozin et al. (2000) Arch. Neurol., 57:793-796).
Accordingly, modulation of amyloid-xcex2 protein export with a modulator of the human ABCB9 transporter would be expected to modulate amyloid deposition and thus, Alzheimer""s disease.
In addition, the ABCB9 transporter molecules of the invention are useful as targets for developing modulating agents of multidrug resistance. Moreover, the molecules of the present invention are useful as diagnostic and therapeutic tools.
Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding ABCB9 transporter proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of ABCB9-encoding nucleic acids.
In one embodiment, an ABCB9 transporter nucleic acid molecule of the invention is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO: 1 or 3, or a complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO: 1 or 3, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO: 3 and nucleotides 1-298 of SEQ ID NO: 1. In another embodiment, the nucleic acid molecule includes SEQ ID NO: 3 and nucleotides 2597-3536 of SEQ ID NO: 1.
In another embodiment, an ABCB9 transporter nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO: 2. In a preferred embodiment, an ABCB9 transporter nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more homologous to entire length of the amino acid sequence of SEQ ID NO: 2.
In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human ABCB9 transporter having the amino acid sequence of SEQ ID NO: 2. In yet another preferred embodiment, the nucleic acid molecule is at least 2298 nucleotides in length. In a further preferred embodiment, the nucleic acid molecule is at least 3536 nucleotides in length and encodes a protein having ABCB9 transporter activity (as described herein).
Another embodiment of the invention features nucleic acid molecules, preferably ABCB9 transporter nucleic acid molecules, which specifically detect ABCB9 transporter nucleic acid molecules relative to nucleic acid molecules encoding non-ABCB9 transporter proteins. For example, in one embodiment, such a nucleic acid molecule is at least 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 500-1000, 1000-1500, 1500-2000, or 2000-2500 or more nucleotides in length and/or hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO: 1, or a complement thereof. It should be understood that the nucleic acid molecule can be of a length within a range having one of the numbers listed above as a lower limit and another number as the upper limit for the number of nucleotides in length, e.g., molecules that are 60-80, 300-1000, or 150-400 nucleotides in length.
In preferred embodiments, the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to nucleotides 299-2596 of SEQ ID NO: 1. In other preferred embodiments, the nucleic acid molecules comprise nucleotides 299-2596 of SEQ ID NO: 1.
In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO: 1 or 3 under stringent conditions.
Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to an ABCB9 transporter nucleic acid molecule, e.g., the coding strand of an ABCB9 transporter nucleic acid molecule.
Another aspect of the invention provides a vector comprising an ABCB9 transporter nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. In yet another embodiment, the invention provides a host cell containing a nucleic acid molecule of the invention. The invention also provides a method for producing a protein, preferably an ABCB9 transporter protein, by culturing in a suitable medium, a host cell, e.g., a mammalian host cell such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.
Another aspect of this invention features isolated or recombinant ABCB9 transporter proteins and polypeptides. In a preferred embodiment, the protein, preferably an ABCB9 transporter protein, includes at least one transmembrane domain and has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more homologous to the amino acid sequence of SEQ ID NO: 2. In yet another preferred embodiment, the protein, preferably an ABCB9 transporter protein, includes at least one transmembrane domain and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1.
In another embodiment, the invention features fragments of the protein having the amino acid sequence of SEQ ID NO: 2, wherein the fragment comprises at least 15 amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ ID NO: 2. In another embodiment, the protein, preferably an ABCB9 transporter protein, has the amino acid sequence of SEQ ID NO: 2.
In another embodiment, the invention features an isolated protein, preferably an ABCB9 transporter protein, which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more homologous to a nucleotide sequence of SEQ ID NO: 1 or 3, or a complement thereof. This invention further features an isolated protein, preferably an ABCB9 transporter protein, which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 3, or a complement thereof.
The proteins of the present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-ABCB9 transporter polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins. The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably ABCB9 transporter proteins. In addition, the ABCB9 transporter proteins, biologically active portions thereof, or expressible nucleic acids encoding the foregoing, can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
In another aspect, the present invention provides a method for detecting the presence of an ABCB9 transporter nucleic acid molecule, protein or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting an ABCB9 transporter nucleic acid molecule, protein or polypeptide such that the presence of an ABCB9 transporter nucleic acid molecule, protein or polypeptide is detected in the biological sample.
In another aspect, the present invention provides a method for detecting the presence of ABCB9 transporter activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of ABCB9 transporter activity such that the presence of ABCB9 transporter activity is detected in the biological sample.
In another aspect, the invention provides a method for modulating ABCB9 transporter activity comprising contacting a cell capable of expressing an ABCB9 transporter with an agent that modulates ABCB9 transporter activity such that ABCB9 transporter activity in the cell is modulated. In one embodiment, the agent inhibits ABCB9 transporter activity. In another embodiment, the agent modulates the ability of the ABCB9 transporter to allosterically modify the function of other membrane proteins. In another embodiment, the agent stimulates ABCB9 transporter activity. In one embodiment, the agent is an antibody that specifically binds to an ABCB9 transporter protein. In another embodiment, the agent modulates expression of ABCB9 transporter by modulating transcription of an ABCB9 transporter gene or translation of an ABCB9 transporter mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an ABCB9 transporter mRNA or an ABCB9 transporter gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant or unwanted ABCB9 transporter protein or nucleic acid expression or activity by administering an agent which is an ABCB9 transporter modulator to the subject. In one embodiment, the ABCB9 transporter modulator is an ABCB9 transporter protein. In another embodiment the ABCB9 transporter modulator is an ABCB9 transporter nucleic acid molecule. In yet another embodiment, the ABCB9 transporter modulator is a polypeptide antibody (or fragment thereof), peptide, peptidomimetic, or other small molecule, e.g. a molecule that is carbohydrate-based, lipid-based, nucleic acid-based, natural organic-based, or synthetically derived organic-based.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding an ABCB9 transporter protein; (ii) mis-regulation of the gene; and (iii) aberrant post-translational modification of an ABCB9 transporter protein, wherein a wild-type form of the gene encodes a protein with an ABCB9 transporter activity.
In another aspect the invention provides a method for identifying a compound that binds to or modulates the activity of an ABCB9 transporter protein, by providing an indicator composition comprising an ABCB9 transporter protein having ABCB9 transporter activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on ABCB9 transporter activity in the indicator composition to identify a compound that modulates the activity of an ABCB9 transporter protein, e.g., an ABCB9 transporter protein associated with a membrane.